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Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal. The annual number of deaths equals the number of newly diagnosed cases, despite maximal treatment. The overall 5-year survival rate of <5% has remained stubbornly unchanged over the last 30 years, despite tremendous efforts in preclinical and clinical science. There is unquestionably an urgent need to further improve our understanding of pancreatic cancer biology, treatment response and relapse, and to identify novel therapeutic targets. Rigorous research in the field has uncovered genetic aberrations that occur during PDAC development and progression. In most cases, PDAC is initiated by oncogenic mutant KRAS, which has been shown to drive pancreatic neoplasia. However, all attempts to target KRAS directly have failed in the clinic and KRAS is widely assumed to be undruggable. This has led to intense efforts to identify druggable critical downstream targets and nodes orchestrated by mutationally activated KRAS. This includes context-specific KRAS effector pathways, synthetic lethal interaction partners and KRAS-driven metabolic changes. Here, we review recent advances in oncogenic KRAS signalling and discuss how these might benefit PDAC treatment in the future.

The development dynamics and self-organization of glandular branched epithelia is of utmost importance for our understanding of diverse processes ranging from normal tissue growth to the growth of cancerous tissues. Using single primary murine pancreatic ductal adenocarcinoma (PDAC) cells embedded in a collagen matrix and adapted media supplementation, we generate organoids that self-organize into highly branched structures displaying a seamless lumen connecting terminal end buds, replicating in vivo PDAC architecture. We identify distinct morphogenesis phases, each characterized by a unique pattern of cell invasion, matrix deformation, protein expression, and respective molecular dependencies. We propose a minimal theoretical model of a branching and proliferating tissue, capturing the dynamics of the first phases. Observing the interaction of morphogenesis, mechanical environment and gene expression in vitro sets a benchmark for the understanding of self-organization processes governing complex organoid structure formation processes and branching morphogenesis. Pancreatic ductal adenocarcinomas (PDAC) exhibit complex morphologies challenging to capture in organoid models. Here, the authors develop PDAC organoids that can recreate branched structures and, with the use of a minimal mathematical model, shed light to pathways and processes directing the dynamics of self-organization and branching morphogenesis.

Background:Although histone deacetylase inhibitors (HDACi) are promising cancer therapeutics regulating proliferation, differentiation and apoptosis, molecular pathways engaged by specific HDAC isoenzymes in cancer are ill defined.Results:In this study we demonstrate that HDAC2 is highly expressed in pancreatic ductal adenocarcinoma (PDAC), especially in undifferentiated tumours. We show that HDAC2, but not HDAC1, confers resistance towards the topoisomerase II inhibitor etoposide in PDAC cells. Correspondingly, the class I selective HDACi valproic acid (VPA) synergises with etoposide to induce apoptosis of PDAC cells. Transcriptome profiling of HDAC2-depleted PDAC cells revealed upregulation of the BH3-only protein NOXA. We show that the epigenetically silenced NOXA gene locus is opened after HDAC2 depletion and that NOXA upregulation is sufficient to sensitise PDAC cells towards etoposide-induced apoptosis.Conclusions:In summary, our data characterise a novel molecular mechanism that links the epigenetic regulator HDAC2 to the regulation of the pro-apoptotic BH3-only protein NOXA in PDAC. Targeting HDAC2 will therefore be a promising strategy to overcome therapeutic resistance of PDAC against chemotherapeutics that induce DNA damage.

Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

Obesity is associated with increased risk for developing pancreatic cancer, and it is suggested that insulin resistance provides the missing link. Here we demonstrate that under the context of genetic susceptibility, a high fat diet (HFD) predisposes mice with oncogenic K-ras activation to accelerated pancreatic intraepithelial neoplasm (PanIN) development. Tumor promotion is closely associated with increased inflammation and abrogation of TNFR1 signaling significantly blocks this process underlining a central role for TNFα in obesity-mediated enhancement of PanIN lesions. Interestingly, however, despite increased TNFα levels, mice remain insulin sensitive. We show that, while aggravating tumor promotion, a HFD exerts dramatic changes in energy metabolism through enhancement of pancreatic exocrine insufficiency, metabolic rates, and expression of genes involved in mitochondrial fatty acid (FA) β-oxidation that collectively contribute to improved glucose tolerance in these mice. While on one hand these findings provide significant evidence that obesity is linked to tumor promotion in the pancreas, on the other it suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause.

Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic Kras(G12D) from the endogenous promoter. We show that primary PDECs are susceptible to Kras(G12D)-driven transformation and form pancreatic ductal adenocarcinomas in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of Kras(G12D) induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease Kras(G12D)-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby explain the crucial role of EGFR for Kras(G12D)-driven carcinogenesis in the pancreas.

Background and aims: Recent studies linked cytokine gene polymorphisms to H pylori related gastric cancer development. The current study evaluated the role of cytokine gene polymorphisms for mucosal cytokine expression, the gastric inflammatory response, and bacterial colonisation during H pylori infection. Patients and methods: In 207 H pylori infected patients with chronic gastritis, polymorphisms at different loci of the interleukin (IL)-10, IL-1B, IL-1 receptor antagonist (IL-1RN), tumour necrosis factor (TNF)-A, and interferon (IFN)-G genes were genotyped by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and allelic discriminating TaqMan PCR. Mucosal cytokine mRNA copy numbers were determined by real time quantitative PCR. Presence of bacterial virulence factors was investigated by cagA, vacAs1/2, and babA2 PCR. Biopsies were assessed with regard to the degrees of granulocytic/lymphocytic infiltration and the presence of intestinal metaplasia (IM) and atrophic gastritis (AG). Results: Proinflammatory IL-1 polymorphisms (IL-1RN*2+/IL-1B−511T/−31C+) were associated with increased IL-1β expression, more severe degrees of inflammation, and an increased prevalence of IM and AG. Carriers of the IL-10−1082G/−819C/−592C alleles (GCC haplotype) had higher mucosal IL-10 mRNA levels than ATA haplotype carriers and were associated with colonisation by more virulent cagA+, vacAs1+, and babA2+ H pylori strains. The TNF-A−307(G/A) and IFN-G+874(A/T) polymorphisms did not influence mucosal cytokine expression or the inflammatory response to H pylori. Conclusions: Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and the long term development of precancerous lesions in H pylori infection. Host polymorphisms are associated with certain bacterial strain types, suggesting host specific colonisation or adaptation. These findings contribute to the understanding of the complex interplay between host and bacterial factors involved in the development of gastric pathology.

Nuclear factor-κB (NF-κB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-κB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-κB activity during S-phase checkpoint activation involving ataxia-telangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on κB response elements. Gene expression analyses revealed that, independent of NF-κB activation in the cytosol, TNF-induced NF-κB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-κB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain- and loss-of function approaches argue that anti-apoptotic NF-κB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.

We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53 R167H and KRAS G12D , and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7–8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease.

Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca 2+ release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. Key messages • Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. • Our aim was to characterize the role of TRPV4 in the control of GI motility. • We found that TRPV4 activation reduced colonic contractility. • Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. • TRPV4 may be a novel pharmacological target in functional GI diseases.